Detection and identification of acid-fast bacilli of Mycobacterium species by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. These methods may take days to 1 or more months. The development of a few highly specific molecular probes for testing cultures growing acid-fast bacilli has greatly reduced the time to identification of some mycobacterial isolates. Recently, the polymerase chain reaction and isothermal nucleic acid amplification techniques have been used in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium tuberculosis in clinical samples. At present, no commercially available amplification assay systems are capable of detecting multiple Mycobacterium species while excluding cross-reactive signals from other bacteria commonly present in clinical samples. We have initiated a new effort to investigate the usefulness of a pressure cycling technique to improve the efficiency of nucleic acid release from mycobacterial cells. The collaboration with Dr. Jang B. Rampal of Beckman Corporation to develop a better endpoint detection method for the 20-some most commonly encountered Mycobacterium species from human samples has continued. Experiments have been successfully performed with a method that simultaneously detects the presence or absence of nucleic acid amplification products from six common clinically isolated Mycobacterium species.